RFLP of 16S-ITSrDNA Region to Differentiate Lactobacilli at Species Level

Yavuz, Elif; Güneş, Hatice; Bulut, Çisem; Harsa, Şebnem; Yenidünya, Ali Fazıl

doi:10.1023/b:wibi.0000043151.09366.d7

Cited by 20 publications

(17 citation statements)

References 13 publications

(12 reference statements)

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“…In the identification of lactobacilli, three restriction enzymes, AluI, HaeIII and TaqI, have been used. Among these enzymes, TaqI has been found to be the most discriminative (Yavuz et al, 2004a). 16S-ITS rRNA gene RFLP with the HaeIII enzyme, on the other hand, has identified both lactococci and enterococci much more efficiently than TaqI (Bulut et al, 2005).…”

Section: Resultsmentioning

confidence: 99%

“…Recently, the 16S-ITS rRNA gene RFLP method has been successfully applied to the species-level identification of lactobacilli (Yavuz et al, 2004a), thermophilic bacilli (Yavuz et al, 2004b) and alkalophilic bacilli (Akbalik et al, 2004). In the identification of lactobacilli, three restriction enzymes, AluI, HaeIII and TaqI, have been used.…”

Section: Resultsmentioning

confidence: 99%

“…The 16S internal transcribed spacer (16S-ITS) rRNA gene RFLP method has already been used for the identification of bacteria of diverse origin: lactic acid bacteria (Bulut et al, 2005), lactobacilli (Yavuz et al, 2004a), thermophilic bacilli (Yavuz et al, 2004b) and alkalophilic bacilli (Akbalik et al, 2004). In the present work, this method was used for the species-level identification of 16 staphylococcal type strains and nine clinical isolates that had been isolated from human and animals.…”

Section: Introductionmentioning

confidence: 99%

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Identification of staphylococci by 16S internal transcribed spacer rRNA gene restriction fragment length polymorphism

Sudağıdan

Yenidünya

Güneş

2005

Journal of Medical Microbiology

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The capacity of 16S internal transcribed spacer (16S-ITS) rRNA gene RFLP to differentiate 16 type strains and nine clinical isolates of staphylococci was evaluated. The 16S rRNA gene was amplified together with the ITS region and the amplification products were digested with TaqI restriction enzyme. Analysis of the 16S-ITS rRNA gene RFLP profiles differentiated each of the 16 type strains into distinct RFLP haplotypes. INTRODUCTIONThe habitat of staphylococci is skin, skin glands and mucous membranes of humans and many animals. The genus Staphylococcus includes both pathogenic and saprophytic strains that have been isolated from animal products such as meat and milk, and from environmental sources such as soil, sea water, fresh water, dust and air samples (Kloos et al., 1991).Coagulase-positive species such as Staphylococcus aureus are the cause of many types of infection (Forbes et al., 1998). During the last two decades, coagulase-negative staphylococci (CNS) have also emerged as pathogens causing medical-device-related infections (von Eiff et al., 2002). Because of the pathogenic potential of these bacteria, effective methods are required for their in vitro identification. Beside fatty acid analyses, many diagnostic tests rely on a miniaturized phenotypic characterization and a set of biochemical reactions. These methods enable the identification of S. aureus isolates, but often fail in the identification of CNS (Stoakes et al., 1994;Renneberg et al., 1995;Martineau et al., 1996). Analysis of specific regions of genomic DNA, on the other hand, has produced much more discriminative data. For example, several genomic targets have been effectively used for the identification of Staphylococcus species, including the 16S rRNA gene (Bialkowska-Hobrzanska et al., 1990;De Buyser et al., 1992), the tRNA gene intergenic spacer (Maes et al., 1997), the internal transcribed spacer (Couto et al., 2001), the heat-shock protein 60 (HSP60) gene (Goh et al., 1996), the chaperonin 60 gene (Goh et al., 1997), the femA gene (Vannuffel et al., 1999), the sodA gene (Poyart et al., 2001), the gap gene (Yugueros et al., 2000) and the nuc gene (Brakstad et al., 1992). Recently, an enterobacterial repetitive intergenic consensus PCR and BOX-PCR were also used in the identification of Staphylococcus epidermidis strains (Wieser & Busse, 2000). . The nine clinical isolates were S. capitis subsp. capitis (n ¼ 2), S. caprae (n ¼ 2), S. cohnii subsp. cohnii (n ¼ 1) and S. epidermidis (n ¼ 4). The clinical isolates were identified by basic phenotypic tests and the Staph ID 32 system (bioMérieux). All strains were cultured in tryptic soy broth (TSB; Merck) at 37 8C and stored in 25 % (v/v) glycerol at À80 8C.16S-ITS rRNA gene PCR. Genomic DNA was isolated as described by Arciola et al. (2001). Briefly, 100 ìl of overnight culture prepared in 5 ml TSB at 37 8C with shaking was pelleted by centrifugation. Cell pellets were resuspended in 45 ìl deionized water and 5 ìl lysostaphin solution (100 ìg ml À1 in dH 2 O; Sigma) and incubated for 10 min...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Identification of staphylococci by 16S internal transcribed spacer rRNA gene restriction fragment length polymorphism

Sudağıdan

Yenidünya

Güneş

2005

Journal of Medical Microbiology

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“…However, Ventura and Zink (2002), using the same conditions as were used in this study, recommend ERIC-PCR as a powerful method for discrimination of L. johnsonii strains. Other authors stated ITS-PCR/RFLP to be suitable for Lactobacilli species identification (Yavuz et al 2004 for strain differentiation and propose to use more discriminating methods.…”

Section: Resultsmentioning

confidence: 98%

Evaluation of biochemical and molecular methods for Lactobacillus reuteri strains differentiation

et al. 2014

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Several biochemical and molecular methods were used for discrimination of four Lactobacillus reuteri strains isolated from goatling and lamb stomach mucosa. Internal transcribed spacer (ITS)-PCR method and protein analysis by SDS-PAGE and MALDI-TOF showed to be suitable for strain discrimination whereas ITS-PCR/RFLP and enterobacterial repetitive intergenic consensus (ERIC)-PCR were not strain specific. The used methods differentiated tested strains into distinct groups; however, the location of strains in groups varied. Consistency in results was observed in the case of L. reuteri E and L. reuteri KO4m that were clustered into the same groups using all techniques, except of MALDI-TOF MS. The last one grouped goatling strains and lamb isolate into separate clusters. All investigated methods, except of ITS-PCR/RFLP and ERIC-PCR, were assessed as appropriate for distinguishing of L. reuteri strains.

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“…Proliferated bacteria in the YM culture medium were collected by centrifugation at 10,000 Â g for 5 min, and DNA templates were prepared by using a Cetyltrimethylammonium bromide (CTAB) method as described by Yavuz et al (2004). Almost the full length of the 16S rRNA gene was amplified with the primers fD1 and rD1 (Weisburg et al 1991).…”

Section: Polymerase Chain Reaction (Pcr) Amplificationmentioning

confidence: 99%

Genetic diversity and spatial distribution of rhizobial bacteria nodulating on black locust (Robinia pseudoacacia) rhizospheres

Shiraishi

Komatsu

Matsushita

et al. 2011

Soil Science and Plant Nutrition

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Black locust (Robinia pseudoacacia L.) is an economically and ecologically important tree in Japan. The species is widely used for afforestation because of its symbiotic relationship with nitrogen-fixing rhizobia. In this study, genetic diversity of rhizobia isolated from black locust nodules and spatial distribution of their genotypes were examined. From a coastal forest, six black locust saplings including the whole root systems were collected and positions of nodules on the roots were recorded. Bacteria were isolated from each nodule and then genotyped by Polymerase Chain Reaction (PCR)-Restriction Fragment Length Polymorphism (RFLP) and sequencing analysis of the 16S rRNA gene. Dendrogram analysis based on PCR-RFLP and sequencing analysis indicated that Mesorhizobium species dominantly colonized black locust roots in this forest, occupying at least 77% of living nodules. Positions and PCR-RFLP genotypes of the nodules revealed that rhizobia isolated from neighbouring nodules tended to have the same genotype in some cases. Statistical analysis has supported this fact. The clustering distribution of nodules in the same RFLP groups should be considered to contribute to the large genetic diversity of rhizobia.

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RFLP of 16S-ITSrDNA Region to Differentiate Lactobacilli at Species Level

Cited by 20 publications

References 13 publications

Identification of staphylococci by 16S internal transcribed spacer rRNA gene restriction fragment length polymorphism

Identification of staphylococci by 16S internal transcribed spacer rRNA gene restriction fragment length polymorphism

Evaluation of biochemical and molecular methods for Lactobacillus reuteri strains differentiation

Genetic diversity and spatial distribution of rhizobial bacteria nodulating on black locust (Robinia pseudoacacia) rhizospheres

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